1. Field of the Invention
The present invention relates to a strain of Escherichia sp. capable of producing O-acetyl homoserine in high yield. More particularly, the present invention relates to a strain of Escherichia sp. capable of producing O-acetyl homoserine in high yield, in which the activity of homoserine acetyl transferase, aspartokinase and homoserine dehydrogenase, in combination with at least one enzyme selected from a group consisting of phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate semi-aldehyde dehydrogenase, are introduced and enhanced. Also, the present invention is concerned with a method of producing O-acetyl homoserine using the strain.
2. Description of the Related Art
Methionine, an essential amino acid for the body, finds a variety of applications in the food and medical industries, such as the use thereof as an additive in animal feed and foods and as a material for parenteral nutrient solutions and medicines. Methionine acts as a precursor for choline (lecithin) and creatine and is used as a material useful for the synthesis of cysteine and taurine. Together with cysteine, methionine is one of two sulfur-containing proteinogenic amino acids. S-Adenosyl methionine, derived from L-methionine, serves as a methyl donor in vivo and is involved in the synthesis of various neurotransmitters in the brain. Methionine and/or S-adenosyl-L-methionine (SAM) is also found to prevent lipid accumulation in the liver and arteries and to alleviate depression, inflammation, liver diseases and muscle pain (Jeon B R et al., J Hepatol., 2001 March; 34(3): 395-401).
As summarized below, methionine and/or S-adenosyl-L-methionine has been thus far known to have the in vivo functions of:
1) suppressing lipid accumulation in arteries and in the liver, where lipid metabolism is mediated, and improving blood circulation in the brain, the heart and the kidneys (J Hepatol. Jeon B R et al., 2001 March; 34(3): 395-401).
2) promoting the digestion, detoxication and excretion of toxic substances and the excretion of heavy metals such as Pb.
3) acting as an antidepressant when methionine is administered in a daily dose of from 800 to 1,600 mg (Am J Clin Nutr. Mischoulon D. et al., 2002 November; 76(5): 1158S-61S)
4) improving liver functions against liver diseases (FASEB J. Mato J M., 2002 January; 16(1): 15-26), particularly, against alcohol-induced liver injury (Cochrane Database Syst Rev., Rambaldi A., 2001; (4): CD002235)
5) showing an anti-inflammatory effect on osteoarthritis and promoting the healing of joints (ACP J Club. Sander O., 2003 January-February; 138(1): 21, J Fam Pract., Soeken K L et al., 2002 May; 51(5): 425-30).
6) acting as an essential nutrient to hair formation and preventing brittle hair and depilation (Audiol Neurootol., Lockwood D S et al., 2000 September-October; 5(5): 263-266).
Methionine for use in animal feed, foods and medicines can be synthesized chemically or biologically.
In the chemical synthesis route, on the whole, methionine is produced through the hydrolysis of 5-(β-methylmercaptoethyl)-hydantoin. However, the synthesized methionine is disadvantageously present in a mixture of L- and D-forms which needs a difficult additional process to separate them from each other. In order to solve this problem, the present inventors developed a biological method for selectively synthesizing L-methionine, a chemical which a patent (WO 2008/103432) has already been applied for. The method, termed in brief “a two-step process”, comprises the fermentative production of an L-methionine precursor and the enzymatic conversion of the L-methionine precursor to L-methionine. The methionine precursor preferably includes O-acetylhomoserine and O-succinyl homoserine. The two-step process is evaluated on terms of having overcome the problems from which the conventional methods suffer, such as sulfide toxicity, feedback regulation in methionine synthesis by methionine and SAMe, and degradation of intermediates by cystathionine gamma synthase, O-succinylhomoserine sulfhydrylase and O-acetylhomoserine sulfhydrylase. Also, compared to the conventional chemical synthesis method of producing DL-methionine, the two-step process has the advantage of being selective for L-methionine only, with the concomitant production of organic acids, such as succinic acid and acetic acid as useful by-products.
Found as an intermediate in the biosynthesis pathway of methionine, O-acetyl-homoserine is used as a precursor for the production of methionine (WO 2008/013432). O-acetyl-homoserine is synthesized from L-homoserine and acetyl-CoA with the aid of O-acetyl transferase as shown in the following formula:L-Homoserine+Acetyl-CoA→O-Acetyl-Homoserine.
In the U.S. patent application Ser. No. 12/062,835 of the present assignee are disclosed a microorganism strain into which a thrA gene responsible for aspartate kinase and homoserine dehydrogenase activity and a Deinococcus-derived metX gene coding for homoserine acetyl transferase are introduced to improve the biosynthesis of L-homoserine and O-acetyl-homoserine, respectively, and a method for producing O-acetyl homoserine at high yield using the same.
In this context, the present inventors conceived that the enhancement of the other three enzymes responsible for the catalytic reactions in the homoserine biosynthesis pathway, that is, phosphoenolpyruvate carboxylase (ppc), aspartate aminotransferase (aspC) and aspartate semi-aldehyde dehydrogenase (asd), would increase a higher production yield of O-acetyl homoserine than would the method of U.S. Ser. No. 12/062,835.
Like the concomitant enhancement of a series of the enzymes involved in the conversion from phosphoenolpyruvate to O-acetylhomoserine according to the present invention, attempts have been made to increase L-amino acid productivity by simultaneously expressing the enzymes which play important roles in the biosynthesis pathways of aspartate-derived L-amino acids, such as L-lysine, L-threonine and L-methionine.
EP00900872 is directed to the effective production of L-lysine, featuring an increase in the activities of a series of enzymes involved in the lysine biosynthesis, including dihydropicolinate synthase (dapA), aspartokinase (lysC), dihydropicolinate reductase (dapB), diaminopimelate dehydrogenase (ddh), tetrahydropicolinate succinylase (dapD), succinyl diaminopimelate diacylase (lysE), aspartate semi-aldehyde dehydrogenase (asd), phosphoenolpyruvate carboxylase (ppc), in E. coli. Japanese Patent Nos. JP2006-520460 and JP2000-244921 describes the effective production of L-theronine in E. coli by increasing the activities of aspartate semi-aldehyde dehydrogenase (asd), phosphoenolpyruvate carboxylase (ppc), aspartokinase (thrA), homoserine dehydrogenase (thrA), homoserine kinase (thrB) and threonine synthase (thrC). Also, WO 2007/012078 discloses a recombinant strain of Corynebacterium capable of producing increased levels of L-methionine in which genes coding for aspartokinase (lysC), homoserine dehydrogenase (hom), homoserine acetyl transferase (metX), O-acetylhomoserine sulfhydrylase (metY), cystathionine gamma synthase (metB), cobalamin-dependent transmethylase (metH); cobalamin-independent methionine synthase (metE), methyltetrahydrofolate reductase (metF), and glucose 6-phosphate dehydrogenase (zwf) are increased in expression level while genes coding for methionine repressor protein (mcbR), homoserine kinase (hsk), S-adenosylmethionine synthetase (metK), and threonine dehydratase (livA) are decreased in expression level.
All of the patents are related to the effective production of aspartate-derived L-amino acids, that is, L-lysine, L-threonine and L-methionine, respectively, featuring the employment of gene combinations depending on the respective products.
In the present invention, a series of enzymes responsible for the catalytic steps from phosphoenolpyruvate to O-acetylhomoserine in the O-acetylhomoserine biosynthesis pathway are designed to be increased in expression level to produce O-acetylhomoserine in higher yield, which has been mentioned nowhere in previous documentation. Further, the enzyme combination employed in the present invention is different from that employed for the production of the aspartate-derived L-amino acid, such as L-lysine, L-threonine or L-methionine, as the final products are different.
Leading to the present invention, intensive and thorough research into the production of O-acetyl homoserine in maximal yield, conducted by the present inventors, resulted in the finding that the concomitant enhancement of the genes encoding aspartate kinase and homoserine dehydrogenase (thrA), and homoserine acetyl transferase (metX) plus a gene encoding at least one enzyme selected from among phosphoenolpyruvate carboxylase (ppc), aspartate aminotransferase (aspC), and aspartate semi-aldehyde dehydrogenase (asd) in the form of a genomic DNA and/or a plasmid in a microorganism strain could bring about a significant increase in the production of O-acetyl homoserine.